Onconase (ONC), which is an amphibian homologue of bovine pancreatic ribonuclease (RNase A), is now undergoing a human clinical trial for the treatment of cancer. ONC is delivered to patients intravenously and is able to kill cancer cells selectively. Hence, ONC has the potential to be the harbinger of a new class of cancer chemotherapeutics based on ribonucleases. Ribonuclease- based chemotherapeutic agents are unrelated to extant chemotherapeutics and thus represent a new stratagem for the clinical treatment of cancer. In contrast to ONC, neither RNase A nor its human homologue-RNase 1-is cytotoxic, despite having 10,000-fold greater catalytic activity than ONC. Moreover, only ONC is immunogenic, and its dosing is limited by renal toxicity. These differential attributes, as well as a desire to understand the biochemical mechanism by which ribonucleases exert specific antitumoral activity, motivate this research proposal. Specific Aims. The five Specific Aims of this research proposal use ideas and methods from organic chemistry, biochemistry, molecular biology, and cell biology to reveal the mechanism by which ribonucleases exert their specific antitumoral activity, and to enhance that activity. During the next grant period, this intent will be achieved in five Specific Aims. Aim 1 is to assess the role of cell- surface charge in ribonuclease cytotoxicity. Aim 2 is to determine the kinetic mechanism of cytosolic entry by ribonucleases. Aim 3 is to create targeted ribonucleases with highly selective toxicity for cancer cells. Aim 4 is to reveal the roles of ribonuclease and its cognate inhibitor protein in vivo. Finally, Aim 5 is to use contrast agents and magnetic resonance imagining to report on ribonucleases in vivo. Significance. The results of the research proposed herein will provide a detailed biochemical understanding of the antitumoral activity of ribonucleases, and could ultimately lead to new cancer chemotherapeutics based on variants of an endogenous human enzyme, RNase 1.